DJ-1 controls T cell differentiation and osteoclastogenesis in rheumatoid arthritis

Herein, we investigated the effect of DJ-1 on helper T cell differentiation, fibroblast-like synoviocyte (FLS) activation, and osteoclastogenesis in rheumatoid arthritis (RA). Serum and synovial fluid (SF) of RA and osteoarthritis (OA) patients were collected, and DJ-1 and H2O2 levels were investigated. CD4+ cells from peripheral blood mononuclear cells (PBMCs) were cultured under type 17 helper T cell (Th17) polarization conditions, and CD4+ T cell differentiation, pro-inflammatory cytokine levels, and soluble receptor activator of nuclear factor kappa-Β ligand (RANKL) were assessed. RA-FLSs were stimulated with 50 μM H2O2, and DJ-1 (10, 50, 100 ng/mL) to evaluate MMP-9, VEGF, TNF-α, and sRANKL production, while RANKL+ FLSs were assessed using flow cytometry. Monocytes were cultured with RANKL or IL-17A with or without DJ-1 and H2O2-pretreated RA-FLS, and tartrate-resistant acid phosphatase (TRAP) staining and RT-qPCR of osteoclast-related genes were performed. The levels of DJ-1 and H2O2 in serum and SF of RA patients were higher than those of OA patients. Under Th17-polarizing conditions, CD4+RANKL+ and CD4+CCR4+CCR6+CXCR3- T cells decreased, whereas CD4+CD25highFoxp3+ T cell increased after DJ-1 administration. Additionally, IL-17A, TNF-α, and sRANKL levels decreased in DJ-1-treated groups. DJ-1 lowered MMP-9, VEGF, TNF-α, and sRANKL levels, and RANKL+ FLS in ROS-stimulated RA-FLS. Both RANKL and IL-17A stimulated osteoclast differentiation, DJ-1 decreased TRAP+ cell count, and the expression levels of TRAP, ATP6v0d2, NFATc1, and CTSK. These findings were also observed in in vitro osteoclastogenesis with DJ-1 pretreated RA-FLS. As DJ-1 regulates Th17/Treg imbalance, pro-inflammatory cytokine production, RA-FLS activation, and osteoclastogenesis, it holds potential for RA therapy.

www.nature.com/scientificreports/ whereas regulatory T cells (Tregs) is decreased in number, and it suppress the autoimmune-mediated process of RA 19,20 . The main effector cells of joint destruction in RA are the FLSs and osteoclasts. Osteoclast differentiation and consequent osteoporosis and joint destruction are more prominent in patients with RA than in healthy controls 21,22 . Osteoclast differentiation is fundamentally induced by receptor activator of NF-κB (RANK)-RANK ligand (RANKL) binding. In RA, RANKL expression of helper T cells and FLSs is upregulated 23,24 ; furthermore, some pro-inflammatory cytokines, such as interleukin (IL)-17A, induce osteoclastogenesis 25 . Therefore, proper management of Th17/Treg imbalance, enhanced osteoclastogenesis, and activated FLS are treatment strategies for RA. DJ-1 a protein comprised of 189 amino acids, is encoded by the PARK7 gene 26 . It was first identified as a protective protein in Parkinson's disease 27 , in which it primarily acts as a ROS scavenger and involved in redox reaction 28 . DJ-1 is expressed in various cells of the central nervous system including astrocytes, microglia, and oligodendrocytes 29 . DJ-1 not only has a neuroprotective role, but also suppresses osteoclast differentiation by scavenging ROS 30 and maintaining metabolic homeostasis of skeletal muscle by modulating the mitochondrial function 31 . Moreover, the extracellular administration of DJ-1 induces angiogenesis and osteogenesis via FGF receptor-1 signaling which implies that DJ-1 not only exert intracellular effect but the extracellular administration of DJ-1 may also have roles in various cells 32 . Furthermore, DJ-1 deficiency-induced Tregs show reduced proliferative potential 33 . Tregs act as self-tolerance inducers and are negatively associated with RA activity 34 . In a study of collagen-induced RA mice, DJ-1-deficient mice showed higher arthritis index and incidence rate than wild-type mice 30 . The aforementioned actions of DJ-1 suggest a potential therapeutic role for DJ-1 in RA progression.
In the present study, we aimed to evaluate the role of DJ-1 in Th cell differentiation. Furthermore, the effect of DJ-1 on RA patients with FLS was assessed. Finally, osteoclastogenesis induced by pro-inflammatory cytokines (IL-17A), RANKL, and RA-FLS was evaluated under various doses of DJ-1.

Materials and methods
Patients. Serum samples were obtained from patients with RA (n = 57) and those with osteoarthritis (OA, n = 30); synovial fluid (SF) samples from patients with RA (n = 30) and those with OA (n = 30) were also collected. The inclusion criteria for patients with RA were as follows: (1) fulfillment of the 2010 classification criteria for RA 35 , and (2) age over 18 years. Peripheral blood was obtained from healthy controls (n = 10). The present study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Informed consent was obtained from all the participants prior to enrolment. The experimental protocol was approved by the Konkuk University Medical Center Human Research Ethics Committee (KUMC 2021-06-007).

Enzyme-linked immunosorbent assay (ELISA) of DJ-1 and ROS measurement in serum and SF.
Briefly, a 96-well plate (Eppendorf, Hamburg, Germany) was coated with monoclonal antibodies against Park7/DJ-1, tumor necrosis factor (TNF)-α, IL-17A, RANKL, IL-6, matrix metalloproteinase (MMP)-9, VEGF (all from R&D Systems, Minneapolis, MN, USA), and IL-1β (PeproTech, Cranbury, NJ, USA) at a concentration of 400 ng/mL and incubated at 4 °C overnight. After blocking with phosphate-buffered saline (PBS)/1% bovine serum albumin /0.05% Tween 20 for 2 h at room temperature (22-25 °C), the test samples and recombinant Park7/DJ-1, TNF-α, IL-17A, RANKL, IL-6, MMP-9, VEGF (all from R&D Systems), and IL-1β (PeproTech) as standards were added to the 96-well plate and incubated at room temperature for another 2 h. The plates were washed four times with PBS and Tween 20 and then incubated with 300 ng/mL biotinylated mouse monoclonal antibodies against Park7/DJ-1, TNF-α, IL-17A, RANKL, IL-6, MMP-9, VEGF (all from R&D Systems), and IL-1β (PeproTech) for 2 h at room temperature. After washing, the streptavidin-alkaline phosphate-horseradish peroxidase conjugate (Sigma, St Louis, MA, USA) was added to the wells for 30 min, followed by another wash and incubation with 1 mg/mL p-nitrophenyl phosphate (Sigma) dissolved in diethanolamine (Sigma) to develop the color reaction. The reaction was stopped by the addition of 1 M NaOH, and the optical density of each well was measured at 405 nm. The lower limit of Park7/DJ-1 was 10 pg/mL. Recombinant human DJ-1 diluted in the culture medium was used as a calibration standard, ranging from 62.5 to 4000 pg/mL. A standard curve was drawn by plotting the optical density against the log of the concentration of the recombinant cytokines, and the curve was used to determine the Park7/DJ-1 concentrations in the test samples.
Similarly, ROS levels were measured using the OxiSelect in vitro ROS/RNS Assay Kit (Cell Biolabs, San Diego, CA, USA). The test sample and the H 2 O 2 standard (Cell Biolabs) were loaded onto a shaded 96-well plate (SPL, Gyeonggi-do, Republic of Korea) under dark conditions, and 1 × catalyst solution was added to each well, followed by incutation at room temperature for 5 min. Then, dichlorodihydrofluorescein (DCFH) solution was added to the wells and incubated for 30 min at room temperature. ROS fluorescence (excitation, 485 nm; emission, 535 nm) was analyzed using a GeminiEM fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA). H 2 O 2 diluted in PBS was used as a calibration standard, ranging from 0.039 to 20 µM.
Co-culture of RA-FLS and osteoclasts. RA-FLSs were pre-stimulated with H 2 O 2 at 50 μM with (10, 50, or 100 ng/mL) or without DJ-1 for 72 h. Human CD14 + monocytes (1 × 10 6 cells/well) were seeded in 24-well plates with α-minimal essential medium and 10% heat-inactivated FBS in the presence of 25 ng/mL rhM-CSF for 72 h. Pre-stimulated RA-FLSs (2 × 10 3 cells/well) were added to the osteoclast culture plate. As a control, non-stimulated RA-FLS-added conditions were used as controls. The medium was supplemented with RANKL (40 ng/mL) and rhM-CSF (30 ng/mL) when RA-FLS was added. The culture medium was changed every 3 days. On days 10-14, TRAP + cells were identified using a TRAP staining kit (Cosmo Bio) 37 .
RNA preparation and quantification of gene expression levels using real-time quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted using an easy-spin Total RNA Extrac- Statistical analysis. All data are expressed as the mean ± standard error of the mean. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni's post-hoc multiple comparison test. Statistical significance was set at P < 0.05. All statistical analyses were performed using Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA).

Serum and SF levels of DJ-1 and H 2 O 2 in patients with RA.
The serum levels of DJ-1 and H 2 O 2 were significantly higher in patients with RA than in those with OA (Fig. 1A). Higher levels of DJ-1 and H 2 O 2 in RA patients were also observed in the SF (Fig. 1B). The serum levels of H 2 O 2 were higher in patients with active RA (DAS28-ESR > 3.2) than in those with inactive disease (DAS28-ESR ≤ 3.2), whereas DJ-1 levels were higher in patients with inactive RA (data not shown).

Effects of DJ-1 on Th differentiation and pro-inflammatory cytokines and soluble RANKL production under Th17-polarizing condition.
In vitro CD4 + cell culture was performed under Th17-polarizing conditions to mimic RA conditions, and the CD4 + cell populations were analyzed using flow cytometry. The gating strategy is shown in Supplementary Fig. S1. Flow cytometry analysis revealed that DJ-1 decreased the www.nature.com/scientificreports/ CD4 + CCR6 + CCR4 + CXCR3 − T cell (Th17) population in a dose-dependent manner. CD4 + IFN-γ + T cells (Th1) and CD4 + RANKL + T cell numbers were suppressed following DJ-1 administration. CD4 + CD25 high Foxp3 + T cell differentiation was significantly increased by DJ-1 treatment in a dose-dependent manner ( Fig. 2A). DJ-1 also suppressed the production of tumor necrosis factor (TNF)-α, IL-17A, and sRANKL in culture media (Fig. 2B).

FLS activation by H 2 O 2 stimulation and inversion by DJ-1 treatment. The demographic, laboratory,
and medication data of RA patients were summarized in Supplementary Table 1. When RA-FLSs were cultured with H 2 O 2, they produced more MMP-9 and sRANKL than the RA-FLSs cultured without H 2 O 2 . DJ-1 administration decreased the H 2 O 2 -induced production of MMP-9 and RANKL in a dose-dependent manner. Further, the levels of VEGF and TNF-α significantly decreased after DJ-1 100 ng/mL administration (Fig. 3A). The gating strategy for this flow cytometric analysis is shown in Fig. 3B. RANKL + FLS were also suppressed in the DJ-1 100 ng/mL administration group (Fig. 3C).

Discussion
In the present study, we demonstrated that the serum and SF levels of DJ-1 and ROS were higher in RA patients than in OA patients. The addition of DJ-1 regulated Th17/Treg imbalance and suppressed the differentiation of RANKL-expressing CD4 + T cells and FLS. In addition, the levels of pro-inflammatory cytokines (TNF-α and IL-17A) and sRANKL production by Th cells were attenuated by DJ-1. ROS-stimulated FLS showed increased MMP-9 and sRANKL secretion, and DJ-1 reversed ROS-induced FLS activation. DJ-1-knockout mice showed higher arthritis scores and incidence rates than those of wild-type mice when an RA-like phenotype was induced by type II collagen immunization (collagen-induced mouse model) 30 ; however, the mechanisms underlying the action of DJ-1 in RA progression were not assessed. In one study, RA-FLS stimulation by oxidative stress increased oxidation protein products and enhanced the gene expression levels of TNF-α, IL-1β, MMPs, and VEGF via the NADPH oxidase-dependent pathway 16 . Regarding T cell regulation by DJ-1, DJ-1 deficiencyinduced Tregs showed a lower replicative and proliferative capacity 33 . In addition, DJ-1-knockout mice showed higher Th1 and Th17 responses and CD3 + T cell migration 38 , which implies a potential role of DJ-1 in T cell differentiation. Regulating the helper T cell subset imbalance is a possible treatment target for RA [39][40][41] ; previous studies have found that upregulation of Th17 and downregulation of Treg (Th17/Treg imbalance) is evident in RA patients 19 . In the present study, DJ-1 administration regulated helper T cell subsets to suppress RA progression (downregulating Th17 and upregulating Treg) and diminished pro-inflammatory cytokine (IL-17A and  www.nature.com/scientificreports/ TNF-α) secretion under Th17-polarizing conditions. Furthermore, TNF-α production was attenuated by DJ-1 administration in vitro following ROS stimulation of RA-FLS. RANKL-and IL-17A-induced osteoclastogenesis were suppressed by DJ-1 treatment. Furthermore, ROSactivated RA-FLS promoted osteoclastogenesis, which was suppressed by adding DJ-1 to the RA-FLS pre-activation state. DJ-1 not only acts in the intracellular space, but can also be secreted 42 . Till date, little is known about the role of extracellular DJ-1. DJ-1 inhibits osteoclastogenesis by blocking intracellular ROS and subsequent TRAF6-and ITAM-mediated signaling pathways 30 . Osteoblasts are known as primary suppliers of RANKL, and the RANK-RANKL interaction is the primary activation signal for osteoclast differentiation and maturation 43 . In RA, other cells such as T cells and FLS can also express RANKL 23,24 , and these cells augment osteoclastogenesis in RA. One of the pivotal pro-inflammatory cytokines in RA pathogenesis is IL-17A, which can promote osteoclastogenesis without RANKL stimulation 25 . We demonstrated that osteoclastogenesis under RANKL and IL-17A stimulation was effectively suppressed by DJ-1 administration. In addition, H 2 O 2 stimulated RA-FLS addition increased osteoclastogenesis, which was suppressed in DJ-1 co-stimulated RA-FLS-added osteoclastogenesis conditions. In addition, the expression of the osteoclast inducing factor, RANKL, was effectively suppressed in both in vitro PBMC culture (under Th17-polarizing condition) and RA-FLS culture (under ROS stimulation) by DJ-1 administration. These results differed from those of a previous study 30 with respect to the method (DJ-1-knockout versus extracellular addition of DJ-1) used and the osteoclast stimulation conditions (IL-17A stimulation). Furthermore, co-culture of osteoclasts and RA-FLS reinforces the idea that DJ-1 exerts both a direct and indirect suppressive role on osteoclastogenesis via regulation of FLS. The present results suggest the potential therapeutic potential of DJ-1 in RA, and the beneficial effects of DJ-1 could also be exerted by external administration.
Rapid and vigorous proliferation of the synovium and pannus formation are the cornerstone pathologic processes of RA 1 , and pannus formation requires higher oxygen delivery. Expression of angiogenetic factors, such as VEGF, is increased in the serum and SF of RA patients 44 , and these angiogenic factors promote neoangiogenesis of the pannus 45 . In addition, the oxidative stress marker of SF positively correlated with the DAS28 of RA patients 46 . This implies that decreasing oxidative stress via an ROS scavenger such as DJ-1 may be a potential therapeutic target in RA treatment. VEGF can induce osteoclastogenesis in RA by dual action: (1) inducing RANKL expression in RA-FLS, and (2) directly inducing osteoclast differentiation in osteoclast precursor cells 47 . Among the various MMPs, MMP-9 promotes RA-FLS survival and cartilage destruction 48 . Furthermore, in a mouse model of RA, MMP-9-knockout mice showed less severe arthritis than wild-type mice 49 , indicating the pivotal role of MMP-9 in RA pathogenesis. In the present study, ROS stimulation increased VEGF and MMP-9 production in RA-FLS, and DJ-1 suppressed ROS-induced VEGF and MMP-9 production in RA-FLS.

Conclusions
In conclusion, we demonstrated the therapeutic potential of DJ-1 in RA pathogenesis. DJ-1 controls the pathologic Th cell subset (Th17, RANKL + CD4 + T cells), induces Treg differentiation, and inhibits pro-inflammatory cytokine production. ROS-stimulated RA-FLS exhibited an attenuated inflammatory response and RANKL/ MMP-9/VEGF expression following DJ-1 treatment. Finally, osteoclastogenesis was attenuated by DJ-1 administration under RANKL/IL-17A stimulated condition. In addition, DJ-1 addition attenuated osteoclastogenesis induced by ROS-stimulated RA-FLS. These findings support the therapeutic potential of DJ-1 exerted via several molecular pathways in RA pathogenesis.